Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof

ABSTRACT

The present invention relates to the use of site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids. Such single-stranded regions can take the form of gaps interior to the duplex, or terminal single-stranded regions. Single-stranded termini can be crafted to allow linkage of various elements via base-pairing with elements containing a complementary single-stranded region. This joining is useful, for example, in an ordered, oriented assembly of DNA modules to create cloning or expression vectors. This joining is also useful in attaching detection probes and purifying DNA molecules containing the single-stranded region. Gaps are useful in similar applications, including attaching detection or purification probes.

BACKGROUND OF THE INVENTION

The joining of DNA fragments is central to the methodology of molecularbiology. There is a consistent need to efficiently join DNA segments toform useful, informative arrays that can allow for analysis in vitro andin vivo. DNA restriction endonucleases, in combination with DNA ligase,have been the principal tools used to create such fragment arrays. Thisapproach relies heavily upon the natural occurrence of appropriaterestriction endonuclease recognition sites, and to a lesser extent onbeing able to insert appropriate sites through such techniques assite-directed mutagenesis, PCR or linker insertion.

The primary method of joining DNA fragments involves enzymatic ligation,preferably with cohesive termini created by restriction endonucleasecleavage such that the two fragments can only be joined in a singleorientation. Often, the product of that ligation is a circular moleculesuitable for transformation into and propagation in a bacterial host.Alternatively, the cohesive termini may be identical, in which case twopossible orientations can result, necessitating screening of the finalproducts. Finally, one or both of the termini may be blunt ends,reducing the efficiency of ligase joining, but also eliminating therequirement for compatible cohesive termini. In a practical sense, thenumber of elements that can be joined is limited to two, possibly threeelements. In addition, in the absence of DNA ligase, no joining isobserved.

One naturally-occurring method that increases the efficiency of fragmentjoining is observed in the life cycle of the bacteriophage lambda. Uponlambda infection, a linear double-stranded genome enters the cell. Thisgenome is circularized prior to replication via pairing of complementary12-nucleotide single-stranded regions at the two ends of the genome.These single-stranded regions are created after replication and prior tophage packaging by the action of the lambda int gene product. The intgene product can be used in vitro in much the same way as restrictionendonucleases to cut and rejoin DNA fragments. In theory, this approachcould be expanded to join multiple elements in a defined, ordered array.However, this would require multiple int-like proteins that recognizedifferent sequences to assure unique orientation of fragments. Thiscould be accomplished by using similar regions from differentbacteriophages (e.g., bacteriophage P22), but this would require theisolation of a separate int gene product for each set of cohesive ends,and potentially introduce difficulties in optimizing reaction conditionsfor more than one cleavage. Additionally, in this approach the cohesivesequence is constrained to the naturally occurring sequence. Thus, whilethis is a possible approach, it is not the optimal approach.

An alternative is to create single-stranded regions by the combinedaction of nucleases. Several methodologies have been described,including: limiting digestion by controlling the time of digestion (Liand Evans Nucleic Acids Res. 15:4165-4166 (1997)), inhibiting digestionat a selected location (Aslanidis and de Jong Nucleic Acids Res.18:6069-6074 (1990); Zhou and Hatahet Nucleic Acids Res. 23:1089-1090(1995); Dietmaier, et al. Nucleic Acids Res. 21:3603-3604 (1993)) andselectively enhancing digestion at a specific location (U.S. Pat. No.5,137,814; Nisson et al. PCR Methods Appl. 1:20-123 (1991)). In stillanother method, a mixture of staggered PCR products are hybridizedtogether to create overhangs (Tillett and Nelian Nucleic Acids Res. 27:e26 (1999)). Most of these methods require a PCR step to add a DNAsequence element or non-standard nucleotide to the termini as a preludeto exonuclease action. The inherent infidelity of PCR raises concerns ofintroducing a mutation into the amplified DNA sequence, thus it would bemore desirable to assemble DNA fragments replicated in vivo.Furthermore, it is difficult to assess whether the desired enzymaticaction has been completed on the DNA termini since the gross propertiesof the fragment (e.g., electrophoretic mobility) are largely unaltered.

SUMMARY OF THE INVENTION

The present invention is related to the production of definedsingle-stranded regions in DNA, and the use of such regions to join,detect and purify such molecules. Site-specific DNA nickingendonucleases are used to form the single-stranded regions by nicking atthe boundaries of the single-strand regions, either on opposing DNAstrands (creating terminal single-stranded regions) or on the samestrand (creating a single-strand gap).

Creation of such single-stranded regions facilitates assembly ofmultiple nucleic acid fragments in an ordered array, either linear orcircular. This is useful in a variety of applications, includingconstruction of vectors with interchangeable cassettes. Although theexamples provided here use the enzyme N.BstNBI, the skilled artisan willappreciate that any other site-specific nicking enzyme would giveequivalent results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Overview of sequence-specific single strand formation usingsite-specific nicking endonucleases.

FIG. 2: Use of N.BstNBI to create 5′ and 3′ cohesive ends andsingle-stranded gaps.

FIG. 3: Examples of applications using DNA molecules withsingle-stranded regions.

FIG. 4: Creating a vector (pAZY2) with two fragments, each flanked byN.BstNBI 18-nucleotide cohesive ends. bla=beta-lactamase gene.Ori=origin of replication.

FIG. 5: Creating an ampicillin-resistance cassette flanked by N.BstNBI18-nucleotide cohesive ends.

FIG. 6: Creating a plasmid (pECH1) from which three fragments can begenerated by N.BstNBI cleavage, each resulting fragment flanked by18-nucleotide cohesive ends.

FIG. 7: Sample scheme to create a family of DNA fragments flanked byidentical N.BstNBI 18-nucleotide cohesive ends.

FIG. 8: Agarose gel analysis of assembly of three fragments with18-nucleotide cohesive ends.

FIG. 9: Creating a plasmid (pECU7) from which three fragments can begenerated by N.BstNBI cleavage, each resulting fragment flanked by12-nucleotide cohesive ends.

FIG. 10: Agarose gel analysis of assembly of three fragments with12-nucleotide cohesive ends.

Oligonucleotide Sequences

216-113 (SEQ ID NO:1) pAAATCAATCTAAAGTATATACCGGTAAACTTGGTCTGACA 216-114(SEQ ID NO:2) pCTAGCATTAGTCAGACTCTACATTCAAATATGTATCCG 216-117 (SEQ IDNO:3) pGCGCTCGATGTCAGACTCGAGCAAAAGGCCAGCAAAAG 216-112 (SEQ ID NO:4)pGAGTCCGATTGACCTAAGCGGATACTCTGACGACTCGTAGAAAAGA TCAAAGGATC 222-14 (SEQID NO:5) pGAGTCTCAGACTATCTGGAGCGACTGACTCAAACTTGGTCTGACAG TTACC 241-83(SEQ ID NO:6) GTAAATATCGGACTCTACAATCAAATATGTATCCGCTCAT 226-10 (SEQ IDNO:7) GATCGAGTCTGACATCGAGCGCCTAGCATTAGTCAGACTCGATATCGAGTCTCAGCCTGTTAGCGATGGTACATGACGACTC 226-11 (SEQ ID NO:8)CTAGGAGTCGTCATGTACCATCGCTAACAGGCTGAGACTCGATATCGAGTCTGACTAATGCTAGGCGCTCGATGTCAGACTC 226-180 (SEQ ID NO:9)CATGTCTAGACTGCAGAGATCT 226-181 (SEQ ID NO:10) AGATCTCTGCAGTCTAGA 235-262(SEQ ID NO:11) TACATTCAAATATGTATCCGC 235-263 (SEQ ID NO:12)TAAACTTGGTCTGACAGTTAC 236-184 (SEQ ID NO:13)GAGTATCCGCTTAGGTCAATCGGACTCGGACCGGATATCACATGTGA GTCGTCA 236-185 (SEQ IDNO:14) CCTGTTAGCGATGGTACATGACGACTCACATGTGATATCCGGTCCGA GTCCGAT 242-11(SEQ ID NO:33) pGAGTCAGCTCAATGTTGCCAGTCAGGACTCGTAGAAAAGATCAAAG GATC242-12 (SEQ ID NO:34) pGGGCCACGTAGACTCGAGCAAAAGGCCAGCAAAAG 244-114 (SEQID NO:50) CTGGCAACATTGATCGGACTCGGACCGGATATCACATGTGAGTCGTCA 244-115 (SEQID NO:51) GCTCCAGATAGTTGACGACTCACATGTGATATCCGGTCCGAGTCCGAT

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to creation of defined single-strandedregions in DNA using site-specific endonucleases that predominantlycleave only one strand of the DNA duplex (nicking endonucleases) atdefined locations. In one embodiment, DNA substrates are designed suchthat the nicking endonuclease(s) makes at least two nicks in duplex DNA.By positioning the sites of these nicks close together, the regionbounded by the nicks in duplex DNA becomes susceptible to dissociation,creating single-stranded regions defined by the cleavage sites. Thecleavage sites are in turn defined by the position of the nicking enzymerecognition site and the disposition of the nicking site relative tothat recognition site.

If the nicking sites occur on the same strand of the DNA duplex, asingle-stranded gap can be created (FIG. 1). If the nicking sites occuron opposite strands of the DNA duplex, dissociation of the strands willcreate complementary single-stranded termini. As described below, bothtypes of single-stranded regions can be useful in a variety ofapplications.

EXAMPLES OF NICKING ENDONUCLEASES

Several types of endonucleases can be envisioned in the context of thepresent invention. The basic requirement is that the enzyme cleave in asite- and strand-specific fashion, acting predominantly on one strand ofthe duplex DNA.

Nicking endonuclease activity has been described for some traditionalrestriction endonucleases. For example, EcoRI endonuclease has beenobserved to nick one strand of its recognition sequence, dissociate andrebind to cleave the other strand, depending on the substrate andreaction conditions (Modrich and Zabel, J. Biol. Chem. 251:5866-5874(1976); Ruben, et al., Nucleic Acids Res. 4:1803-1813 (1977); Rubin andModrich, Nucleic Acids Res. 5:2991-2997 (1978)). However, there is noknown strand specificity for the nicking, a possible consequence of thesymmetry of the recognition site. Additionally, cleavage of the secondstrand of the nicked recognition site occurs with similar kinetics ascleavage of the first strand, eventually converting all nicked sites todouble-strand cleavages. Thus, while this class of enzyme could producenicked species for use in the present invention, such enzymes are notthe preferred enzymes.

Proposals have been tendered for conversion of double-strandedendonucleases into enzymes that can cut only a single strand of the DNAduplex (e.g., Waugh and Sauer, Proc. Natl. Acad. Sci. USA 90:9596-9600(1993); Jeltsch, et al., Trends Biotechnol. 14:235-238 (1996)). Enzymesso converted continue to recognize a specific sequence, but cleave onlyone strand, or predominantly one strand of duplex DNA. For those enzymeswith symmetric recognition sequences, this class of altered enzymeswould suffer from the same limitations as described above forrestriction endonucleases. However, if enzymes with asymmetricrecognition sequences can be induced to nick predominantly or entirelyin one strand such enzymes would be useful in the present invention.Conversion of restriction endonucleases into site-specific nickingenzymes has been described (Stahl, et al., Proc. Natl. Acad. Sci. USA93:6175-6180 (1996); Bitinaite, et al., Proc. Natl. Acad. Sci. USA95:10570-10575 (1998)). However, these references do not describe theuse of such enzymes in the creation of single-stranded regions.

Chemical modification of the DNA substrate has been used to limitrestriction endonuclease action to site- and strand-specific nicking.For example, restriction endonucleases have been identified that nick atrecognition sites in which one of the scissile phosphodiester bonds hasbeen replaced by a phosphorothioate linkage (Taylor, et al., NucleicAcids Res., 13:8749-8764 (1985)). This methodology has been used inconjunction with exonuclease III digestion to create large, primarilyrandom regions for oligonucleotide-directed mutagenesis (Taylor, et al.,Nucleic Acids Res. 13:8765-8785 (1985)) and to create polymerase entrysites in strand displacement amplification (Walker, et al., Proc. Natl.Acad. Sci. USA 89:392-396 (1992)), but not to create single-strandedregions with specific borders as provided in the present invention.However, with appropriate positioning of the phosphorothioate linkages,and judicious selection of restriction endonucleases, nicks couldpotentially be introduced allowing implementation of the presentinvention. However, this is not the preferred way to practice thepresent invention due to the added step of introducing thephosphorothioate linkage.

Elements of replication machinery with site-specific nicking activityhave also been described. For example, Horiuchi, et. al. have studiedthe nicking reaction catalyzed by the filamentous bacteriophage f1 gpII,a reaction that is involved in initiation of rolling circle DNAreplication of the phage genome (Higashitani, et al., J. Mol. Biol.237:388-400 (1994)). As such, the site- and strand-specific nickingproperties of this enzyme could potentially be harnessed for use in thepresent invention.

While the above described enzymes and methods for introducing a site-and strand-specific nick in duplex DNA can be exploited in the presentinvention, a more preferred nicking enzyme is exemplified by N.BstNBIendonuclease (New England Biolabs, Beverly, Mass.) or its isoschizomerN.BstSEI (Abdurashitov, et al., Mol. Biol. (Mosk) 30:1261-1267 (1996)).These enzymes have characteristics of being elements of restrictionmodification systems, namely the endonuclease is accompanied by asite-specific methyl transferase that modifies the recognition sequenceand blocks cleavage by the endonuclease. The recognition site forN.BstNBI is: 5′ . . . GAGTCNNNN^(∇)N . . . 3′ (SEQ ID NO:15) 3′ . . .CTCAGNNNN N . . . 5′where the cleavage site is indicated by “^(∇)” and N indicates any base(subject to the normal rules of base pairing between the strands).Importantly, the N.BstNBI recognition sequence is asymmetric, otherwiseno strand bias would be expected.

By analogy with the wide variety of restriction endonucleases that havebeen discovered and characterized, a reasonable expectation is thatadditional nicking enzymes of this type will be identified, some ofwhich will act on different recognition sequences. Site-specific nickingendonucleases can be identified during routine screening for restrictionendonucleases, as was N.BstNBI. This identification relies on theoccurrence of two closely-disposed recognition sites, each sitedirecting nicking on opposite strands of the DNA substrate. In such anarrangement, a double-strand cleavage event is scored. Determination ofthe sites of cleavage, combined with analysis of the sequencessurrounding the cleavage sites, allows a determination of therecognition site for the cleavage activity. Because restrictionendonuclease cleavage sites generally leave no more than a 4-nucleotidecohesive end, traditional screening does not need to take steps topromote dissociation of cohesive ends. Thus, site-specific nickingendonucleases are only identified in this methodology when sites areclose enough to produce short cohesive ends. Identification may beimproved by post-cleavage treatment of the DNA to promote dissociationof longer cohesive ends, such as heating the sample to 65° C. prior togel analysis. Newly-identified site-specific nicking endonucleases maycut to one side of the recognition site, as with N.BstNBI, or possiblyinternal to the recognition sequence. Both of these types of nickingenzymes would be functional in the present invention by correctlydisposing the corresponding nicking enzyme recognition site about thetarget region.

The Disposition of Nicking Sites Determines the Character of theSingle-Stranded Region

Placement of the nicking enzyme recognition sequence is a crucialelement in determining the nature of the single-stranded region that iscreated. As discussed above, the disposition of nicking sites can giverise to single-stranded gaps or to cohesive termini. The use ofsite-specific nicking enzymes to produce those nicks necessitatesplacing the corresponding recognition sites near the nicking sites,placing restrictions on the sequences in and/or around thesingle-stranded region. The nature of these constraints is dictated bythe disposition of the nicking site relative to the recognition site andon whether a gap or single-stranded termini is generated. For example,if the nicking site is internal to the recognition sequence, thenelements of the recognition sequence inevitably reside in both thesingle-strand sequence created and in the flanking sequences. As aconsequence, some flexibility is lost in designing unique cohesivetermini. When, however, the nicking site is located outside therecognition sequence, three configurations are possible, each withdesirable features. First, recognition sites can be oriented so thatcleavage occurs between the two sites (FIG. 2 a). As the recognitionsites are outside of the single-stranded region, there are no a prioriconstraints on this region. Sequences on both sides of the cohesivetermini are constrained to having the recognition sequence. Second, bothsites can be oriented so that cleavage occurs exterior to the two sites(FIG. 2 b). In this configuration the flanking sequences are notconstrained, although the single-stranded region must include both ofthe two recognition sequences. Finally, both sites can be oriented intandem, with one site within the single-stranded region and the otheroutside, with the net result being two nicks on the same strand (FIG. 2c). In this configuration, sequences to one side of and within thesingle-stranded region are constrained. The third arrangement will notproduce cohesive ends, but rather a gapped single-strand portion of DNA.

While the illustration envisions using an enzyme that nicks to the 3′end of the recognition sequence, enzymes that cleave 5′ of therecognition sequence should give similar results, with the differencebeing creation of 3′ instead of 5′ overhangs with the first scenario,and 5′ instead of 3′ overhangs with the second scenario.

Additionally, although the illustration displays the use of twocleavages to create the single-stranded regions, more nicking sites maybe clustered to create multiple nicks on the same strand of the DNAduplex. This may be desirable, for example, to produce smallsingle-strand fragments that will be more easily dissociated to exposegaps or single-strand termini.

The illustration also shows use of a single nicking enzyme to performnicking reactions. Two separate nicking enzymes could also beappropriately mixed to generate a similar product. Alternatively,single-stranded termini could be created by introduction of a singlenick near the terminus of a linear molecule, where the linear moleculeeither exists naturally, is a synthetic molecule, or is created by invitro reactions such as, but not limited to, restriction endonucleasecleavage or the polymerase chain reaction (PCR).

The sequence of the single-strand cohesive ends can be defined by theuser, as illustrated in the accompanying example. The distance betweenthe nicking sites will determine the length of the cohesive region. Foruse in joining DNA fragments, the length of this region between nicksshould be (1) long enough to allow unique, stable pairing betweenelements under the reaction conditions used in the joining reaction and(2) short enough to allow the single-stranded cohesive ends to beseparated by denaturing forces that leave the rest of the DNA intactduring the isolation steps preceding joining. The exact reactionconditions defined by these limitations depend, at least, ontemperature, ionic strength, and DNA concentration. The examplesillustrated below employ cohesive ends 12 or 18 nucleotides long,although lengths both shorter and longer are also in the scope of thepresent invention.

Design of Extended Single-Strand Cohesive Ends:

One advantage of the described invention is the ability to design thesequence of the cohesive ends formed during the nicking reaction. Whilenot absolutely required, ideally, these cohesive ends will be able toefficiently anneal only with their complements. Several characteristicsare described to help ensure this pairing. The cohesive ends ideally:

-   -   1) Should have little propensity to form secondary structure.        Secondary structure has the potential to create self-pairing        that competes with the desired pairing with the complementary        strand.    -   2) Should have minimal ability to form a complementary duplex        with themselves or any of the cohesive ends in the assembly        mixture other than the designed pair.

Other characteristics are also desirable, but not absolutely required,for the cohesive ends:

-   -   3) Have a GC content of about 50% to maximize the combinations        possible and normalize the predicted melting temperature of the        various annealed cohesive ends.    -   4) Lack restriction sites with recognition sites larger than 4        base pairs. This is a minor point that potentially gives more        flexibility in having unique restriction sites in final        constructs.

Many protocols could yield sequences that fit the above design criteria,including one briefly outlined below. The skilled artisan willappreciate that it is the design characteristics rather than rigidadherence to design protocols that is central to the present invention.For example, sequences do not necessarily need to be randomly generatedand sequence alignments and secondary structure predictions can befashioned using alternate software or manual manipulations.

-   -   1) Generate a random nucleotide sequence in which:        |G−C|=0 or 1   a.        |A−T|=0 or 1   b.        |(G+C)−(A+T)|=0 or 1   c.        where G, C, A and T represent the number of G, C, A and T        residues, respectively.    -   2) Using the GCG (Genetics Computer Group, Inc., Madison, Wis.)        software program foldRNA (Chan, et al., Nucleic Acids Res.        19:353-358 (1991)), check for potential secondary structure of        the single strand. Bias selection to those with unfavorable        folding energies.    -   3) Using the GCG program mapsort (Genetics Computer Group, Inc.,        Madison, Wis.), check for the occurrence of restriction sites,        particularly those for the site-specific nicking endonuclease to        be employed, or with recognition sequences longer than four        nucleotides.    -   4) Using the GCG (Genetics Computer Group, Inc., Madison, Wis.)        program GAP (Needleman and Wunsch, J. Mol. Biol. 48:443-453        (1970)), check for self-hybridization and for predicted        hybridization between all possible sets of single-strand ends        (the sequence itself and its complement). Note the number of        matched base pairs in the alignment, the number of consecutive        base pairs, and the length of the gap between paired        nucleotides. Also note whether the predicted pairing creates a        nicked junction or a gap. Bias the selection to those with few        matched base pairs, short patches of consecutive base pairs and        with maximal gaps between paired nucleotides.    -   5) Select from the pool of generated sequences those that best        fit the above criteria.

One possible limitation on this construction is the occurrence ofrecognition sites for the site-specific nicking endonuclease within theDNA fragment, thus producing nicks beyond those required to create thegaps or cohesive ends. Several approaches are possible to circumventthis limitation. (1) Sequence elements can be chosen that lack suchsites. (2) Sites existing within the element can be eliminated bydeletion or mutation. Where the site exists in coding regions, thismutation can be designed to be translationally silent, and thus notaffect the coding capacity of the gene. (3) If the nicking sites are farenough apart, dissociation of the individual internal pieces can berestrained by choice of reaction conditions. The skilled artisan willrecognize that these conditions will depend on the length of thesingle-stranded region and on the GC content within this region, in bothcases relative to the portions of the DNA that will remaindouble-stranded following treatment. Parameters that promotesingle-strand formation include, but are not limited to, elevatedtemperatures, low ionic strength and alkaline pH. These parameters canbe manipulated individually or in combination to give the desiredresult, namely dissociation of the single-stranded region withoutdissociation of the remaining portions of the DNA duplex. Appropriateconditions can be deduced by incrementally varying the above parametersand observing the nature of the products, for example by gelelectrophoresis as described in the examples. The mildest conditionsable to effect single-strand formation are preferable in the presentinvention. As such conditions are identified, nicks within the elementsthat are far enough apart will be transparent in the cloning protocolsnoted below. (4) Finally, as multiple nicking enzymes are identified,judicious choice of enzymes can reduce the potential for cleavage withinthe coding regions.

Creation and Use of Cassette Creation Vectors:

In order to be useful in assembling DNA fragments, the gene fragmentsmust be flanked by appropriately disposed cleavage sites. The skilledartisan will recognize that these sequences can be added in a number ofways, giving the same final product. For example, synthetic ornaturally-occurring oligonucleotide duplexes containing correctlydisposed nicking sites and associated sequences can be ligated to theends of a desired fragment. Alternatively, PCR primers can be designedwith priming sequences preceded by 5′ tails containing correctlydisposed nicking sites, thus adding the appropriate sequences during thePCR reaction. Site-directed mutagenesis can also be used to introducesites in desired locations. Whatever the method of attachingappropriately disposed sites, the operation of the single-strandedregions is expected to be the same.

Alternatively, the desired DNA fragment can be inserted betweenpre-existing flanking sequences. Successful insertion and growth of theplasmid allows cleavage and isolation of the fragment with appropriatesingle-stranded cohesive termini flanking the inserted fragment (FIG.7). One utility of such a cassette creation vector is the ability toaccumulate a family of DNA fragments, each bounded by identical cohesivetermini. These could be useful as interchangeable parts, for example inan expression vector. Multiple families of such elements would allowrapid assembly of DNA arrays, for example containing a desiredreplication origin, a selective marker, promoters, enhancers andassociated control elements, allowing constitutive and regulated geneexpression, auxiliary factors (such as repressors and activators, andinhibitors), and a gene or DNA sequence of interest.

Use of Cohesive Ends Created by Site-Specific Nicking Enzymes:

The creation of single-stranded regions can be useful in a variety ofapplications. First, and as documented in the examples, these regionscan be used for oriented assembly of multiple DNA fragments, either intolinear arrays or into circular molecules. Such concatenates are useful,for example, in assembly of cloning vectors, placing inserts intocloning vectors, assembling DNA fragments of discrete molecular weightsto be used as molecular weight standards in DNA analysis, and in joininggene coding regions to transcriptional and translational controlelements to allow expression of the gene coding region.

It is worth noting that the present invention provides methods forcreating cohesive DNA termini of virtually any composition, and as suchtermini can be created to be complementary to cohesive ends created byrestriction endonucleases. This provides a means for meshing thistechnology with pre-existing cloning systems that utilize cohesive ends.Fragments generated by the present invention can thus be joined to thosecreated by restriction endonucleases, homing endonucleases or enzymessuch as lambda int.

The ability to selectively expose single-stranded regions in duplex DNAis useful in a variety of applications. For example, exposing suchregions allows selective mutagenesis of such exposed regions bysingle-strand specific mutagenesis such as bisulfite. Single-strandedgaps are also preferred over double-stranded regions as binding sitesfor oligonucleotides, such as those that might be employed foroligonucleotide-directed mutagenesis (Zoller and Smith, DNA 3:479-488(1984)).

Single-stranded regions, whether internal gaps or terminal cohesiveends, can serve as annealing sites for probes, providing a means forsequence-specific detection of the exposed region, and in turn theattached DNA fragment. For example, DNA fragments could be encoded withunique sequence tags that could be revealed as single-strand regionsusing the present invention. DNA fragments bearing a given code can thenbe identified by annealing the code complement, bearing a detectionreagent, to the family of DNA fragments. Only the desired DNA fragmentwill then be illuminated. Detection reagents might include radioactive,fluorescent, or antigenic moieties attached to or incorporated into theprobe.

The single-strand regions exposed via the present invention can also beused in a purification scheme to isolate specific fragments of DNA. TheDNA fragment containing the exposed single-stranded region is passedover a solid support to which has been affixed a single-stranded DNAcontaining a sequence complementary to that exposed region, or a portionthereof. The skilled artisan will recognize that there are conditions ofsalt, buffer and temperature at which only the complementary sequenceswill bind to the solid matrix, and those not binding can be washed off.Elution of the desired fragment can be accomplished by changing thebinding conditions, for example by raising the binding temperature,preferably in a buffer of low ionic strength, to release the bound DNA.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

The references cited above and below are herein incorporated byreference.

EXAMPLES

While several of the following examples were established with the enzymeN.BstNBI, any sequence-specific nicking enzyme may be substituted inaccordance with the present invention. The sequence listings in thebelow plasmids are most often inferred from the sequence of the originalplasmids, and do not reflect possible errors introduced duringconstruction, for example by replication errors during PCR.

EXAMPLE 1 Generation of Three 18-Nucleotide Cohesive Sequences

To test the effectiveness of creating and using N.BstNBI-derivedcohesive ends in cloning, three such termini were devised and created. Anumber of random sequences were generated that matched the criteriadetailed above. To that end, the following steps were taken:

-   -   1) Generate a random 18-nucleotide sequence from a pool of four        G, five A, five C and four T residues.    -   2) Use the GCG (Genetics Computer Group, Inc., Madison, Wis.)        program foldRNA (Chan, et al., Nucleic Acids Res. 19:353-358        (1991)) to calculate the free energy for the most stable        conformer of the single strand. Give preference to those with        the most unfavorable folding energies.    -   3) Using the GCG program Mapsort (Genetics Computer Group, Inc.,        Madison, Wis.), check for the occurrence of restriction enzyme        recognition sites (within the overlap and contiguous sequences),        particularly for that of N.BstNBI or for those of enzymes with        recognition sequences longer than four nucleotides. Give        preference to those sequences lacking restriction sites, taking        into account flanking contiguous sequences.    -   4) Using the GCG (Genetics Computer Group, Inc., Madison, Wis.)        program GAP (Needleman and Wunsch, J. Mol. Biol. 48:443-453        (1970)), check for self-hybridization and for predicted        hybridization between all possible sets of single-strand ends        (the sequence itself and its complement). Note the number of        Watson-Crick base pairs in the match, the number of consecutive        base pairs, and the length of the gap between paired        nucleotides. Also note whether the predicted pairing creates a        nicked junction or a gap. Give preference to those with minimal        predicted base pairing, and with gaps created in the most stable        minimal base pairing scheme.    -   5) When restriction sites are apparent, or when particular base        pairing interferes with desired patterns, swap nucleotides        within the sequence and test again.

The cohesive ends generated in this example were selected from thosebest fitting the above criteria from a limited pool of randomlygenerated sequences. Table 1 lists 18 nucleotide sequences analyzedusing this protocol, with the first sequence in each of the seven setsbeing the initially generated sequence, and subsequent entries beingvariants of that sequence designed to better conform to the establishedcriteria. One column also reports the free energy calculated for thegiven sequence (The underlined sequences are those actually used in thelater Examples). TABLE 1 Generated 18 nucleotide regions, with predictedfolding energy calculated by the GCG program foldRNA. Sequence energy I.GCGTCTAAACCCAGATGT (aaa) −3.5 (SEQ ID NO:16) GCGTTCAAACCCAGATGT (aab)−0.5 (SEQ ID NO:17) II. AGCTGTTCTAAGCCGCAA (aac) −0.8 (SEQ ID NO:18)III. TGTGAACACCTCGTAACG (aad) −0.6 (SEQ ID NO:19) IV. TTCCCAAGCACATGGGAT(aae) −5.6 (SEQ ID NO:20) TCTCCAAGCACAGTGAGT (aaf) 0.3 (SEQ ID NO:21) V.TGACTCAAGCGAGTACTC (aag) −0.2 (SEQ ID NO:22) TGACTCAAGCGGATACTC (aah)1.5 (SEQ ID NO:23) TGACCTAAGCGGATACTC (aaz) 1.5 (SEQ ID NO:24) VI.ACTGAGCGCCATGCATTA (aai) −1.2 (SEQ ID NO:25) ACTGAGCGCCAGTCATTA (aaj)1.0 (SEQ ID NO:26) ATCGAGCGCCATGCATTA (aak) 1.0 (SEQ ID NO:27)ATCGAGCGCCTAGCATTA (aal) 1.0 (SEQ ID NO:28) VII. TGTACCATCGCTAACAGG(aay) 0.4 (SEQ ID NO:29)

TABLE 2 GCG GAP Analysis of test pairs aaf aaz aal aay aaf 18/18/0 rev 8/3/0 aaz  7/3/2 18/18/0 rev  6/2/1  6/2/3 aal  7/5/1  6/4/3 18/18/0rev  6/3/8  7/3/1  6/1/1 aay  8/2/0  7/3/1  6/3/1 18/18/0 rev  7/3/0 6/4/1  6/3/6  6/2/0

The indicated oligonucleotides, as listed in Table 1, were processedpairwise using the GCG program GAP. Numbers record results derived fromthe program output. The first number is the total number of base pairsformed, the second number indicates the longest series of consecutivebase pairs among those pairs, and the final number indicates the lengthof the gap predicted at the terminus. For example, the output of aazpaired with aal is: 1 TGACCTAAGCGGATACTC . . . 18 (aaz) (SEQ ID NO:24)         ||||  || 1 . . . ATCGAGCGCCTAGCATTA 18 (aal) (SEQ ID NO:28)displaying six base pairs, with four consecutive base pairs and a gap ofthree nucleotides at the terminus. Lines preceded by “rev” record valuesfor the complement of the indicated sequence.

The three most favorable sequences among those tested were aaz, aal andaay. These were framed within N.BstNBI sites to create, after cleavage(indicated by ^(∇) or _(Δ)), 18-nucleotide cohesive ends: 5′GAGTCTGAC^(∇)ATCGAGCGCCTAGCATTA GTCAGACTC 3′ N1      (SEQ ID NO:30) 3′CTCAGACTG TAGCTCGCGGATCGTAAT_(Δ)CAGTCTGAG 5′ N1′ 5′GAGTCCGAT^(∇)TGACCTAAGCGGATACT CTGACGACTC 3′ N2      (SEQ ID NO:31) 3′CTCAGGCTA ACTGGATTCGCCTATGAG_(Δ)ACTGCTGAG 5′ N2′ 5′GAGTCTCAG^(∇)CCTGTTAGCGATGGTACA TGACGACTC 3′ N3      (SEQ ID NO:32) 3′CTCAGAGTC GGACAATCGCTACCATGT_(Δ)ACTGCTGAG 5′ N3′

As a naming convention, the 18-nucleotide cohesive ends were designated“N” with a number indicating a unique end. A prime symbol (′) indicatesthe complementary strand.

EXAMPLE 2 Creation of Vector Cassettes with Cohesive Termini

A plasmid was created that could generate individual origin andampicillin resistance elements flanked by N.BstNBI elements disposed tocreate joinable 18-nucleotide cohesive ends. The plasmid was created byligating together two PCR fragments, one containing the replicationorigin from pUC19, and the other containing the ampicillin resistancegene, again from pUC19. The primers used for PCR contained extrasequence elements, including appropriately disposed N.BstNBI recognitionsites and sequences to create 18-nucleotide cohesive sequences fittingthe criteria outlined above. All oligonucleotides used in this exampleand others described in the present invention were from New EnglandBiolabs Organic Synthesis Division (Beverly, Mass.).

PCR was performed to generate fragments as follows:

Replication origin cassette: 1×NEB Thermopol buffer, 0.1 mg/ml bovineserum albumen, 0.4 mM dNTPs, 0.5 μM primer 216-112 (SEQ ID NO:4), 0.5 μMprimer 216-117 (SEQ ID NO:3), 1 μg/ml pUC19 DNA (New England Biolabs,Beverly, Mass.), 4 mM MgSO₄ in a final volume of 0.5 ml. Five Units ofVent® DNA polymerase (New England Biolabs, Beverly, Mass.) were added,and the sample was then heated to 94° C. for 3 minutes, followed by 20rounds of thermocycling: 94° C. (15 seconds), 58° C. (15 seconds), 72°C. (60 seconds). The sample was then phenol extracted and ethanolprecipitated. The sample was suspended in gel-loading buffer and run ona 1% agarose gel in TBE. The correct size band was excised and elutedusing an Elutrap® elution chamber (Schleicher & Schuell, Keene, N.H.) at200 volts in 0.5×TBE for two hours. The eluted sample was collected,extracted with phenol and ethanol precipitated.

Ampicillin resistance cassette: 1×NEB Thermopol buffer, 0.1 mg/ml bovineserum albumen, 0.4 mM dNTPs, 0.5 μM primer 216-113 (SEQ ID NO:1), 0.5 μMprimer 216-114 (SEQ ID NO:2), 1 μg/ml pUC19 DNA (New England Biolabs,Beverly, Mass.), 8 mM MgSO₄ in a final volume of 0.5 ml. Five Units ofVent® DNA polymerase (New England Biolabs, Beverly, Mass.) were added,and the sample was then heated to 94° C. for 3 minutes, followed by 20rounds of thermocycling: 94° C. (15 seconds), 58° C. (15 seconds), 72°C. (60 seconds). The sample was then phenol extracted and ethanolprecipitated. The sample was suspended in gel-loading buffer and run ona 1% agarose gel in TBE. The correct size band was excised and elutedusing an Elutrap® elution chamber (Schleicher & Schuell, Keene, N.H.) at200 volts in 0.5×TBE for two hours. The eluted sample was collected,extracted with phenol and ethanol precipitated.

The desired plasmid (FIG. 4) was created by ligating the above mentionedfragments, mixing approximately 10 ng of the replication origin PCRproduct with 30 ng of the ampicillin resistance PCR product in 1×NEBuffer T4 DNA ligase in a final volume of 20 μl. 400 Units of T4 DNAligase (New England Biolabs, Beverly, Mass.) were added, and thereaction incubated at 16° C. overnight. Aliquots of the ligationreaction were transformed (The NEB Transcript 6:7 (1994)) into strainER2502, and ampicillin-resistant colonies were screened for formation ofa circular plasmid containing both elements oriented such that thedirection of transcription for ampicillin resistance was the same as thedirection of replication from the origin element. One such plasmid waspAZY2. This plasmid was transformed into strain ER2502, withtransformants selected by growth on LB-ampicillin plates at 37° C. Asingle colony was used to inoculate 0.5 liter of LB (per liter, 10 gtryptone, 5 g yeast extract, 10 g NaCl, 1 g dextrose, 1 g MgSO₄.6H₂O, pHadjusted to 7.2 with NaOH) containing 0.1 mg/ml ampicillin, and thesample was grown overnight with shaking at 37° C. Plasmid DNA wasisolated from the resulting culture with a Qiagen Mega-prep (Qiagen,Inc., Chatsworth, Calif.) as directed by the supplier. The concentrationof the closed circular pAZY2 was determined by spectrophotometry.

The individual cassettes containing the origin of replication andampicillin resistance elements were formed by cleavage of purified pAZY2by N.BstNBI (New England Biolabs, Inc., Beverly, Mass.) using reactionconditions suggested by the supplier. Following cleavage by N.BstNBI,samples were heated to 75° C. for 20 minutes to dissociate the cohesiveends, and immediately loaded on a 1.4% agarose gel in 1×TBE buffer,where the fragments were separated by electrophoresis. The replicationorigin and ampicillin resistance cassettes were separately eluted in anElutrap® elution chamber (Schleicher & Schuell, Keene, N.H.) for 2 hoursin 0.5×TBE at 200 volts. Samples were phenol extracted, ethanolprecipitated, dissolved in TE and quantified by running aliquots on agel along with mass standards. The mass of DNA fragments wasextrapolated by visual inspection.

EXAMPLE 3 Vector Reassembly with Two DNA Fragments Containing 18-bpCohesive Ends

The ability of the DNA fragments containing 18-bp cohesive termini toefficiently reassemble was tested by mixing together the separatedampicillin resistance and replication origin cassettes described inExample 2, and scoring reassembly by transformation. Assembly reactionscontained 1× NEBuffer T4 DNA ligase and approximately 0.9 pM DNA(equivalent to 1 μg/ml of pAZY2). Samples contained either (1)covalently closed circular pAZY2, (2) the approximately 704 bp N.BstNBIfragment from pAZY2 containing the pUC19 origin of replication, (3) theapproximately 1021 bp N.BstNBI fragment from pAZY2 containing theampicillin resistance gene from pUC19, or (4) an equimolar mixture ofthe replication origin and ampicillin resistance gene fragments. 10 μlsamples of each were incubated at room temperature for one hour alone orin the presence of 200 units of T4 DNA ligase (New England Biolabs,Beverly, Mass.).

After incubation, 1 μl of each assembly reaction was mixed with 50 μl ofcompetent Escherichia coli cells (The NEB Transcript 6:7 (1994)) strainER2502 on ice for 10 minutes. The samples were then placed in a 37° C.water bath for 45 seconds, and incubated on ice for 10 minutes. Cellsamples were then diluted by addition of 1 ml of LB liquid media, and0.1 ml of each diluted sample was plated onto LB agar plates containing0.1 mg/ml ampicillin. Plates were incubated overnight at 37° C., and thenumber of colonies then determined. TABLE 3 Transformation of a two-partre-assembly of pAZY2. The heading “ccc pAZY2” indicates covalentlyclosed circular pAZY2; “ori” the N.BstNBI fragment derived from pAZY2containing the pUC19 origin of replication; “amp^(R)” the N.BstNBIfragment derived from pAZY2 containing the pUC19 ampicillin resistancegene. Entries with numbers separated by a semi-colon are from replicatetrials. ccc pAZY2 ori amp^(R) T4 DNA ligase # colonies + + 67 + 93;128 + +  0 +  0; 0 + +  0 +  0; 0 + + + 26 + + 19; 37

As seen in Table 3, no transformants were observed with the isolatedorigin of replication or ampicillin resistance DNA fragments alone. Thenumber of colonies observed after assembly showed an efficientreassembly, with mixtures of the fragments yielding between 20-40% thenumber of colonies observed with the parent circular pAZY2 DNA. In theseexperiments, addition of T4 DNA ligase did not significantly change thenumber of transformants observed.

EXAMPLE 4 Creation and Isolation of Cassettes Flanked by CohesiveTermini

For some applications it may be desirable to have a family ofinterchangeable DNA fragments, each flanked by a common set of cohesivetermini. To facilitate the creation of such DNA fragments, a cloningvector was created in which a cloning site, in this case a unique EcoRVsite, was situated between two pairs of correctly disposed N.BstNBIsites. After insertion of the DNA fragment of interest into the EcoRVsite, in this example the ampicillin resistance gene, cleavage withN.BstNBI liberated this cloned gene cassette with 18-nucleotide cohesivetermini.

The cloning vector in this example, pEBE1, was created via a multi-stepassembly process (FIG. 5). The backbone for pEBE1 is the plasmid pNB0, aderivative of Litmus 28 (New England Biolabs, Beverly, Mass.) thatcontains no N.BstNBI sites and was created in the laboratory of HuiminKong of New England Biolabs. Briefly, pNB0 was created usingsite-directed mutagenesis to alter the N.BstNBI sites within thebeta-lactamase gene (coordinate 931) and replication origin region(coordinate 1939), while successive deletion of the SwaI/DraIII andSnaBI/PvuII fragments removed the remaining N.BstNBI sites. The pNB0construct was cut with BspLU11I and HpaI and ligated to anoligonucleotide duplex of oligonucleotides 226-180 (SEQ ID NO:9) and226-181 (SEQ ID NO:10), adding BglII and XbaI sites to the plasmid. Theresulting plasmid was named pEBA5. The BglII/XbaI-cleaved pEBA5 wasligated to an oligonucleotide duplex (oligonucleotides 226-10/226-11;SEQ ID NO:7-8), adding two pairs of inwardly-directed N.BstNBI sitesseparated by an EcoRV site (vector pEBE1). This plasmid is thusconfigured to place a restriction fragment between two pairs of N.BstNBIsites, and upon treatment with N.BstNBI the liberated insert is flankedby unique 18-nucleotide cohesive ends, corresponding to N1 and N3 above.

An ampicillin resistance cassette was created by ligating a PCR productderived from pUC19 to pEBE1 cut with EcoRV. The PCR reactions wereperformed in 0.1 ml reaction mixtures containing 1×NEB Thermopol buffer,0.1 mg/ml bovine serum albumen, 0.4 mM dNTPs, 1 μg/ml pUC19 DNA, 0.5 μMprimer 235-262 (SEQ ID NO:11), 0.5 μM primer 235-263 (SEQ ID NO:12), 1unit of Vent® DNA polymerase (New England Biolabs, Beverly, Mass.) andvarying concentrations of MgSO₄ (4, 6, 8, 10 or 12 mM). PCR conditionswere 94° C. for three minutes, then 20 cycles of 94° C. (15 seconds),58° C. (15 seconds) and 72° C. (60 seconds). Following amplification,aliquots of each of the five reactions were run on an agarose gel andfound to create an equivalent yield of the expected approximately 1 kbfragment. All samples were pooled, extracted with a phenol/chloroformmixture and ethanol precipitated. The precipitated sample was suspendedin TE buffer (10 mM TrisHCl (pH 8.0), 1 mM EDTA), and subjected toagarose gel electrophoresis to further purify the product. The separatedband was localized by UV fluorescence in the presence of ethidiumbromide, and electroeluted at 200 volts for 2.5 hours in an Elutrap®apparatus using conditions specified by the manufacturer (Schleicher &Schuell, Keene, N.H.). The eluted sample was ethanol precipitated andsuspended in TE buffer. This purified fragment was ligated to pEBE1 cutwith EcoRV using standard conditions. One successful ligation productcontaining an ampicillin resistance gene transcribed in the samedirection as plasmid replication was named pECF11. Cleavage of thisconstruct with N.BstNBI, followed by heating at 65° C. for five minutes,liberated an ampicillin cassette flanked by 18-nucleotide cohesive ends.

EXAMPLE 5 Construction of Plasmid Vector pECH1

A plasmid from which three fragments could be generated by N.BstNBIcleavage, each with two 18-nucleotide cohesive ends, was created byligating a synthetic DNA duplex of oligonucleotides 236-184 (SEQ IDNO:13) and 236-185 (SEQ ID NO:14), with the replication origin N.BstNBIfragment isolated from pAZY2 and the ampicillin resistance N.BstNBIfragment from pECF11, the latter two fragments isolated as describedabove. The oligonucleotide duplex was annealed by mixing 1 μM of eachsingle strand in 0.2 ml annealing buffer (20 mM TrisHCl (pH 7.6), 50 mMNaCl, 10 mM MgCl₂) in a 1.5 ml eppendorf tube. This mixture was floatedin a 400 ml beaker filled with 95° C. water, and the beaker left out atroom temperature until the temperature of the water was below 30° C. Inthis assembly, all three fragments have complementary 18-nucleotidecohesive ends, permitting a unique, oriented assembly of the fragments.

The assembly was accomplished by incubating approximately 4 ng of theorigin-containing fragment, 6 ng of the ampicillin resistance-containingfragment, and 0.3 ng of the oligonucleotide duplex in 10 μl of NEBufferT4 DNA ligase at room temperature for 1 hour. Following this incubation,2 μl of the assembly mixture was transformed into ER2502 as describedabove, and transformants selected by growth on LB-ampicillin plates at37° C. One desired construct was named pECG1. pECG1 was further modifiedby cutting with RsrII (New England Biolabs, Beverly, Mass.) and PciI(SibEnzymes, Novosibirsk, Russia), and ligating this vector to anapproximately 3.2 kb DNA fragment isolated from lambda DNA aftercleavage with RsrII and PciI. The resulting construct was named pECH1.Digestion with N.BstNBI resulted in three DNA fragments, approximately3.2, 1.0 and 0.7 kb in length.

EXAMPLE 6 Vector Reassembly with Three DNA Fragments Containing18-Nucleotide Cohesive Termini

Vector assembly was repeated, similar to that described in Example 3,but using three fragments produced by N.BstNBI cleavage (Example 4) ofpECH1. Cleavage of pECH1 by N.BstNBI resulted in the three fragmentsdescribed in Example 5. The digested N.BstNBI fragments weredrop-dialyzed on 0.025 μM nitrocellulose filters (Millipore Corporation,Bedford, Mass.) against TE, then heated to 65° C. for 3 minutes.Fragments were then separated by electrophoresis on a ¹% agarose gel in0.5×TBE, with both the gel and buffer pre-heated to 65° C. The fragmentswere excised and eluted using an Elutrap® elution chamber (Schleicher &Schuell, Keene, N.H.) at 200 volts in 0.5×TBE for 3 hours. The elutedsamples were collected, extracted with phenol and ethanol precipitated.Finally, the samples were suspended in TE buffer and quantified byvisual inspection after running aliquots alongside DNA mass standards onan agarose gel.

In this case, DNA assembly was performed at a concentration of about 0.3nM of each fragment, corresponding to about 1 μg/ml of pECH1. Assemblywas performed in NEBuffer N.BstNBI (10 mM TrisHCl (pH 7.5 at 25° C.), 10mM MgCl₂, 0.15 M KCl, 1 mM dithiothreitol) at room temperature for 1hour. From the assembly reactions, 1 μl aliquots were transformed into50 μl aliquots of ER2502 using standard protocols, and diluted into 1 mlLB broth. Samples (0.3 ml) of the diluted transformants were plated onLB-ampicillin plates and grown overnight at 37° C. No transformants wereobserved in assembly reactions containing a single DNA fragment, or anytwo of the fragments. When all three fragments were present, 16 colonieswere observed, compared with 126 colonies obtained with the covalentlyclosed circular pECH1 DNA. This corresponds to a transformationefficiency of 5.3×10⁴ cfu/ug for the assembled pECH1 and 4.2×10⁵ cfu/ugof covalently closed circular pECH1.

Reassembly was also visualized by mixing these three fragments andobserving the assembled products by gel electrophoresis. Arrayscontaining 15 ng of each fragment in 10 μl of 1× NEBuffer N.BstNBI weremixed, and incubated at room temperature for 30 minutes, then separatedby electrophoresis on a 1% agarose gel in 1×TBE buffer along with MWstandards from New England Biolabs (FIG. 8). The individual fragmentsran as single bands, while pairwise combinations yielded an additionalband of the expected molecular weight. Combinations with the largerfragment (3.2 kb) were largely depleted for this fragment, as would beexpected since its molar concentration was 3-4-fold lower than theorigin of replication or ampicillin resistance fragment. Mixing theorigin of replication and ampicillin resistance fragments led toformation of the expected hybrid, although not all fragments wereassembled. Finally, mixtures of all three fragments gave a series ofhigher molecular weight species, presumably reflecting linear, relaxedopen circular molecules, or concatamers thereof.

EXAMPLE 7 Vector Reassembly with Four DNA Fragments

The plasmid pECH1 was also used as a source of DNA fragments to evaluatea four-part assembly of fragments, in this case where one of the joiningjunctures is the three-nucleotide overhang generated by SapI. The 3.2 kbN.BstNBI fragment from pECH1 was cut with SapI, yielding two fragmentsof approximately 1.4 and 1.8 kb. Assembly reactions of 20 μl contained1× NEBuffer N.BstNBI with approximately 2 nM of four DNA fragmentsderived from pECH1, namely the three fragments described in Example 6,with the 3.2 kb fragment additionally cut by SapI. After incubation atroom temperature for 1 hour, the volume of the reaction was increased to30 μl by addition of ATP (final concentration 0.67 mM) and T4 DNA ligase(final concentration 13 U/μl; New England Biolabs, Beverly, Mass.).After mixing, this sample was incubated at 16° C. for 2 hours. Aliquotsof this ligated mixture were transformed into 50 μl aliquots ofcompetent ER2502 cells, which were in turn plated on LB-ampicillin mediaand incubated overnight at 37° C. to select for transformants.Equivalent quantities of covalently closed circular pECH1 weretransformed in parallel. TABLE 4 Transformation of a four-partreassembly of pECH1. The heading “Closed circular” represents uncleavedcovalently closed circular pECH1; “Reassembled” represents the assemblyof the four fragments derived from pECH1 by cleavage with N.BstNBI andSapI, as described in Example 7. Colony forming units = cfu. ClosedCircular Reassembled Trial 1 9.4 × 10⁵ cfu/μg 9.1 × 10⁵ cfu/μg Trial 22.1 × 10⁵ cfu/μg 9.6 × 10⁴ cfu/μg Trial 3 2.5 × 10⁵ cfu/μg 1.8 × 10⁵cfu/μg

EXAMPLE 8 Generation of 12-Bp Cohesive End Sequences:

In theory, the length of the cohesive ends could be nearly any value. Atthe two extremes, too short a region limits the base pairing andstacking interactions that support duplex formation, and joining is notstable. On the other hand, too large a region leaves the cleaved ends sostable that separation and purification of the DNA fragments isdifficult. The examples above have used 18-nucleotide cohesive ends. Forcomparison, and to demonstrate the universality of the approach,ligation assemblies were also performed with 12-nucleotide cohesiveends, again generated by N.BstNBI.

The methodology of Example 1 was used to generate 12-nucleotide cohesiveends. Procedures were the same, except the randomized sequence was 12nucleotides in length rather than 18. Table 5 lists 12 randomlygenerated sequences along with their predicted RNA folding energies.Table 6 takes a subset of these sequences and compares predicted, moststable pairing configurations for a subset of these sequences. TABLE 5Generated 12 nucleotide regions, with predicted folding energycalculated by the GCG program foldRNA. Sequence foldRNA energyGTAGTTACGCCA (baa) (SEQ ID:35) 2.4 CAATGTTGCCAG (bab) (SEQ ID:36) 4.1TCTCAATGAGGC (bac) (SEQ ID:37) 1.4 AGCGAGCCTTTA (bad) (SEQ ID:38) 2.7TGATCGAGACCT (bae) (SEQ ID:39) 5.9 TCTGCGGATAAC (baf) (SEQ ID:40) 3.6TATGCAGCGCAT (bag) (SEQ ID:41) 1.3 GATCGAACGTTC (bah) (SEQ ID:42) 1.8AAATTTGGGCCC (bai) (SEQ ID:43) 2.0 ACTATCTGGAGC (baj) (SEQ ID:44) 2.6AGGCGACATTTC (bak) (SEQ ID:45) 2.8 ATTTACGGGCCA (bal) (SEQ ID:46) 5.9

TABLE 6 GCG GAP Analysis of test pairs, 12-nucleotide. The indicatedoligonucleotides, as listed in Table 5, were processed pairwise usingthe GCG program GAP. Numbers record results derived from the programoutput. The first number is the total number of base pairs formed, thesecond number indicates the longest series of consecutive base pairsamong those pairs, and the final number indicates the length of the gappredicted at the terminus. baa bab bad bae baf baj bak bal baa . . . rev12/12/0  6/2/1 bab . . . rev  6/4/1 12/12/0  4/2/0  6/3/4 bad . . . rev 4/2/2  4/3/2 12/12/0  5/3/1  5/2/1  4/2/1 bae . . . rev  4/2/0  4/2/0 7/4/2 12/12/0  4/2/1  5/2/2  5/2/0  6/2/0 baf . . . rev  4/2/3  4/2/1 4/2/2  4/2/1 12/12/0  5/4/3  5/3/1  4/2/1  5/3/1  6/2/2 baj . . . rev 4/1/2  5/2/1  4/4/5  6/3/1  5/3/2 12/12/0  4/3/6  4/2/1  4/2/1  6/3/1 7/4/0  4/2/0 bak . . . rev  4/2/4  5/2/3  6/3/6  5/3/1  4/2/2  3/2/612/12/0  7/4/0  8/5/1  5/4/3  5/3/6  3/2/4  5/2/2  6/2/4 bal . . . rev 6/4/2  4/2/3  5/3/3  5/2/1  6/3/1  5/2/0  3/1/2 12/12/0  4/2/2  5/3/3 5/2/2  5/2/0  5/2/0  4/3/6  4/1/1  4/1/1

The three most favorable sequences among those tested were bab (SEQ IDNO:36), bal (SEQ ID NO:46) and baj (SEQ ID NO:44). These were framedwithin N.BstNBI sites to create, after cleavage (indicated by ^(∇) or_(Δ)), 12-nucleotide cohesive ends: 5′ GAGTCAGCT^(∇)CAATGTTGCCAGTCAGGACTC 3′ M1    (SEQ ID NO:47) 3′ CTCAGTCGA GTTACAACGGTC_(Δ)AGTCCTGAG5′ M1′ 5′ GAGTCCGAT^(∇)ATTTACGGGCCA CGTAGACTC 3′ M2    (SEQ ID NO:48) 3′CTCAGGCTA TAAATGCCCGGT_(Δ)GCATCTGAG 5′ M2′ 5′ GAGTCTCAG^(∇)ACTATCTGGAGCGACTGACTC 3′ M3    (SEQ ID NO:49) 3′ CTCAGAGTC TGATAGACCTCG_(Δ)CTGACTGAG5′ M3′

As a naming convention, the 12-nucleotide cohesive ends were designated“M” with a number indicating a unique end. A prime symbol (′) indicatesthe complementary strand.

EXAMPLE 9 Creation of Vector Cassettes with 12-nucleotide CohesiveTermini

A plasmid was created that could generate the individual origin andampicillin resistance elements flanked by N.BstNBI elements disposed tocreate joinable 12-nucleotide cohesive ends. The end result, pECU7 (FIG.9), is analogous to pECG1, differing primarily in the length of cohesivetermini created by N.BstNBI cleavage. As a starting point in theconstruction, a plasmid was created by ligating together two PCRfragments, one containing the replication origin from pUC19, and theother containing the ampicillin resistance gene, again from pUC19. Theprimers used for PCR contained extra sequence elements includingappropriately disposed N.BstNBI recognition sites and sequences tocreate 12-nucleotide cohesive termini fitting the criteria outlinedabove, and allowing unique, oriented reassembly.

PCR was performed to generate fragments as follows:

Replication origin cassette: 1×NEB Thermopol buffer, 0.1 mg/ml bovineserum albumen, 0.4 mM dNTPs, 0.5 μM primer 242-11 (SEQ ID NO:33), 0.5 μMprimer 242-12 (SEQ ID NO:34), 0.1 μg/ml pUC19 DNA (New England Biolabs,Beverly, Mass.), 4 mM MgSO₄ in a final volume of 0.1 ml. One unit ofVent® DNA polymerase (New England Biolabs, Beverly, Mass.) was added,and the sample was then heated to 94° C. for 4 minutes, followed by 20rounds of thermocycling: 94° C. (15 seconds), 58° C. (15 seconds), 72°C. (60 seconds). The sample was then phenol extracted and ethanolprecipitated. The sample was suspended in gel-loading buffer and run ona 1% agarose gel in TBE buffer. The correct size band was excised andeluted using an Elutrap® elution chamber (Schleicher & Schuell, Keene,N.H.) at 200 volts in 0.5×TBE for 1.3 hours. The eluted sample wascollected, extracted with phenol and ethanol precipitated. Finally, thesample was suspended in TE buffer.

Ampicillin resistance cassette: Reaction mixtures contained 1×NEBThermopol buffer, 0.1 mg/ml bovine serum albumen, 0.4 mM dNTPs, 0.5 μMprimer 222-14 (SEQ ID NO:5), 0.5 μM primer 241-83 (SEQ ID NO:6), 1 μg/mlpUC19 DNA (New England Biolabs, Beverly, Mass.) and either 0, 2, 4, 6 or8 mM added MgSO₄, each in a final volume of 0.1 ml. One Unit of Vent®DNA polymerase (New England Biolabs, Beverly, Mass.) was added to each,and the samples were then heated to 94° C. for 3 minutes, followed by 20rounds of thermocycling: 94° C. (15 seconds), 58° C. (15 seconds), 72°C. (60 seconds). Aliquots of the separate reactions were run on anagarose gel to assess the reaction products. The most complete products,from samples containing 2, 4, 6 or 8 mM added MgSO₄, were pooled, andpurified by gel electrophoresis on a 1.4% agarose gel run in TBE buffer.The correct-sized band was excised and eluted using an Elutrap® elutionchamber (Schleicher & Schuell, Keene, N.H.) at 200 volts in 0.5×TBE for1.5 hours. The eluted sample was collected, extracted with phenol,ethanol precipitated and suspended in TE buffer. Approximately 2 μg ofthe purified fragment in a volume of 9 μl was mixed with 1 μl of 10×NEBuffer for T4 DNA ligase, and phosphorylated by addition of 5 units ofpolynucleotide kinase (New England Biolabs, Beverly, Mass.) andincubated at 37° C. for 45 minutes.

The desired plasmid was created by ligating the above mentionedfragments, mixing approximately 0.1 μg of the replication origin PCRproduct with 0.3 μg of the ampicillin resistance PCR product in 1×NEBuffer T4 DNA ligase in a final volume of 20 μl. 2000 Units of T4 DNAligase (New England Biolabs, Beverly, Mass.) were added, and thereaction incubated at 16° C. overnight. Aliquots of the ligationreaction were transformed (The NEB Transcript 6:7 (1994)) into strainER2502, and ampicillin-resistant colonies were screened for formation ofa circular plasmid containing both elements oriented such that thedirection of transcription for ampicillin resistance was the same as thedirection of replication from the origin element. One such plasmid waspECL3 (FIG. 9). This plasmid was transformed into strain ER2502, withtransformants selected by growth on LB-ampicillin plates at 37° C. Asingle colony was used to inoculate 0.5 liter of LB containing 0.1 mg/mlampicillin, and the sample grown overnight with shaking at 37° C.Plasmid DNA was isolated from the resulting culture with a QiagenMega-prep (Qiagen, Inc., Chatsworth, Calif.) as directed by thesupplier. The concentration of pECL3 was determined by visuallycomparing UV fluorescence of aliquots of the samples relative to DNAmass standards run on an agarose gel.

The plasmid pECL3 was modified to add RsrII and PciI restriction sitesbetween the adjacent N.BstNBI sites, allowing insertion of a “stufferfragment.” The plasmid pECL3 was digested with N.BstNBI, andapproximately 25 ng of the digested plasmid was mixed with approximately0.75 pmole of the DNA duplex prepared by annealing oligonucleotides244-114 (SEQ ID NO:50) and 244-115 (SEQ ID NO:51; annealed as describedin Example 5) and 2.5 μl of 10× NEBuffer T4 DNA ligase in a final volumeof 25 μl. This sample was placed in a 70° C. water bath for five minutesto separate the 12-nucleotide cohesive ends in pECL3, then cooled atroom temperature for 35 minutes. The oligonucleotide duplex wasconstructed to contain complementary cohesive ends that annealed tocohesive termini on the ampicillin and replication origin fragmentscreated by N.BstNBI cleavage and selective denaturation of pECL3, andthus the reannealing generated a population of clones that contained thesubstituted oligonucleotide duplex. One such construct was named pECU7(FIG. 9).

An approximately 3.2 kb fragment, derived via RsrII and PciI cleavage oflambda DNA (see Example 5), was ligated to pECU7 cleaved with the sametwo restriction endonucleases, yielding a construct pECV1, similar topECH1.

EXAMPLE 10 Vector Reassembly with Three DNA Fragments Containing12-Nucleotide Cohesive Ends

Vector assembly was repeated, similar to that described in Example 6,but using three fragments produced by N.BstNBI cleavage of pECV1 (seeExample 9). Vector pECV1 was digested with N.BstNBI, followed by dropdialysis of the fragments on a 0.025 μM nitrocellulose filter (MilliporeCorporation, Bedford, Mass.) against TE buffer. NaCl was added to bringthe salt concentration to 30 mM, and the digested DNA was heated to 65°C. for 5 minutes before electrophoresis on a 1% agarose gel in 0.5×TBE.Both the gel and buffer were pre-heated to 62° C. The three fragments,approximately 3.2, 1.0 and 0.7 kb, were excised from the gel and elutedin an Elutrap® elution chamber (Schleicher & Schuell, Keene, N.H.) for 3hours in 0.5×TBE at 200 volts. Samples were ethanol precipitated,dissolved in TE and quantified by agarose gel electrophoresis alongsideDNA mass standards.

In this case, DNA assembly was performed at a concentration of about 3nM each fragment, corresponding to 10 μg/mi of pECV1. Assembly wasperformed in TE augmented with 0.15 M KCl at room temperature for 1hour. From the assembly reactions, 1, 3 or 5 μl aliquots (correspondingto about 10, 30 or 50 ng DNA) were transformed into 50 μl aliquots ofER2502 using standard protocols (The NEB Transcript 6:7 (1994)), anddiluted to 0.2 ml with LB broth; the diluted transformation mixtureswere plated on LB-ampicillin plates and incubated over the weekend atroom temperature. As a control, about 5 ng of covalently closed circularpECV1 was transformed into 50 μl ER2502 cells and afterward, thetransformation mixture was diluted to 1.0 ml with LB broth. Aliquots(20, 60 and 100 μl) of the diluted transformants were plated onLB-ampicillin plates and grown over the weekend at room temperature. Notransformants were observed in assembly reactions containing a singleDNA fragment, or any two of the fragments. When all three fragments werepresent, an average of 3.5×10³ cfu/μg was observed, as compared with3.4×10⁵ cfu/μg in the control covalently closed circular pECV1.

Reassembly was also visualized by mixing these three fragments, andobserving the assembled products by gel electrophoresis. Arrayscontaining about 15-25 ng of each fragment in 10 μl of 1× NEBufferN.BstNBI were mixed, and incubated at room temperature for 60 minutes,then separated by electrophoresis on a 1% agarose gel in 1×TBE bufferalong with MW standards from New England Biolabs (FIG. 10). Theindividual fragments ran as single bands, while pairwise combinationsyielded an additional band of the expected molecular weight, althoughnot all fragments were assembled. Mixtures of all three fragments gave aseries of higher molecular weight species, presumably reflecting linear,relaxed open circular molecules, or concatamers thereof.

EXAMPLE 11 Vector Reassembly with Four DNA Fragments

Plasmid pECV1 was also used to generate DNA fragments for reassemblytests using four fragments. Similar to Example 7, this assembly includedthree N.BstNBI junctions (with 12-nucleotide overhangs) plus one SapIjunction consisting of three-nucleotide overhangs. The SapI junction wasderived from cleavage of the 3.2 kb N.BstNBI fragment from pECV1 withSapI, a process that yielded two fragments of approximately 1.4 and 1.8kb. Assembly reactions of 8.7 μl contained 0.12 M KCl with approximately1.5 nM of four DNA fragments derived from pECV1, namely the threefragments described in Example 10, with the 3.2 kb fragment additionallycut by SapI. After incubation at room temperature for 1 hour, the volumeof the reaction was increased to 20 μl by the addition of ATP (finalconcentration 1 mM), MgCl₂, (final concentration 10 mM), DTT (finalconcentration 10 mM) and T4 DNA ligase (final concentration 20 U/μl; NewEngland Biolabs, Beverly, Mass.). After mixing gently, this reactionmixture was incubated at 16° C. for 2 hours. Aliquots (2, 6 and 10 μl)of the ligation mixture, corresponding to about 10, 30 and 50 ng of DNA,were transformed into 50 μl ER2502 cells; LB media was added to thetransformation mixtures, to a final volume of 0.2 ml and thetransformants were plated on LB-ampicillin media and incubated overnightat 37° C. As a control, 5 ng of covalently closed circular pECV1 wastransformed into 50 μl of ER2502 cells; the transformation mixture wasdiluted to 1.0 ml with LB broth and aliquots of 20 μl (about 0.1 ng), 60μl (about 0.3 ng) or 100 μl (about 0.5 ng) were plated on LB-ampicillinplates which were then incubated overnight at 37° C. An average of9.7×10³ cfu/μg were seen in the 4-way reassembled pECV transformationscompared with 1.5×10⁵ cfu/μg of covalently closed circular pECV1.

1-21. (cancelled) Add new claims 22-29 as-filed in the ParentApplication Ser. No. 09/738,444 on Apr. 19, 2002 in Applicants ResponseA.
 22. A nucleic acid molecule having at least one recognition site andan associated nicking site for a site specific single strand nickingendonuclease, the nucleic acid further comprising a target single strandregion in double-stranded DNA.
 23. A nucleic acid molecule according toclaim 22, having a first and a second single strand nicking site whereinthe first nicking site is orientated in tandem with respect to thesecond nicking site.
 24. A nucleic acid molecule according to claim 22,having a first and optionally a second single strand nicking site suchthat the number of nucleotides between the first and the second nickingsite or the first nicking site and a terminus of the nucleic acidmolecule determines the length of a single-stranded region in the targetsingle stranded region created.
 25. A nucleic acid molecule according toclaim 22 having a first single-stranded target region, the first regionforming a stable pairing with a second single stranded target region ofDNA having a complementary sequence.
 26. A nucleic acid moleculeaccording to claim 24, wherein the single-stranded region has a lengthin the range of 12 to 18 nucleotides.
 28. A nucleic acid moleculeaccording to claim 27, wherein the single-stranded region has a sequenceselected from any of SEQ ID NO:16 through SEQ ID NO:32.
 29. A nucleicacid molecule according to claim 27, wherein the single-stranded regionhas a sequence selected from any of SEQ ID NO:35 through SEQ ID NO:49.